Javid Ghaemmaghami

Multicellular organisms develop and maintain differentiated cell states through a combination of cell-autonomous and non-autonomous regulatory mechanisms, including signal transduction pathways mediated by ligands, receptors, and kinases. Genetic perturbation through CRISPR pooled screening is widely used to study cell-autonomous regulatory mechanisms, but the challenges of cellular-resolution in situ measurement have limited the study of non-autonomous regulatory mechanisms. We developed Perturb-Tags, a spatial multi-omic technique for pooled CRISPR screening that tags nuclei with spatial barcodes, allowing joint measurement of single-nucleus gene expression, CRISPR guide RNAs, chromatin accessibility, and spatial positions within intact tissue. Perturb-Tags achieves cellular-resolution, unbiased gene expression and multi-omic profiling, and higher sensitivity than previous spatial perturbation approaches. We used Perturb-Tags to systematically perturb ligands, receptors, and all expressed kinases in human embryoid bodies. These experiments revealed extensive non-autonomous gene expression changes after perturbation, including distance-dependent loss of signaling pathway activity upon ligand knockdown, compensatory upregulation of alternative signaling pathways, and responses to receptor and kinase knockdown that vary with cell microenvironment. We also identified changes in the spatial organization of cells with ligand and kinase knockdowns. Finally, we trained a graph neural network to model the combined effects of genetic perturbations on gene expression and chromatin accessibility across an entire tissue. Our work provides a powerful experimental and computational platform for mechanistic studies of non-autonomous regulatory mechanisms across healthy and diseased tissues.

Importance: Preterm survivors of necrotizing enterocolitis (NEC) face substantial neurodevelopmental impairment, but the regional biology and timing of associated brain abnormalities remain poorly defined. Objective: To define the regional, cellular, molecular, and temporal features of NEC-associated brain pathology. Design: Prospective multimodal clinical cohort study (2012–2024), complemented by postmortem tissue analyses. Setting: Level IV neonatal intensive care unit at Children’s National Hospital, Washington, DC. Participants: Of 400 infants screened, 253 preterm infants born before 37 weeks’ gestation (128 with NEC and 125 without NEC) were included across neuroimaging, plasma metabolomic, and postmortem cohorts. Exposures: Medical or surgical NEC (modified Bell stage ≥II). Main Outcomes and Measures: Primary outcomes were term-equivalent magnetic resonance imaging (MRI) abnormalities and serial ultrasound hindbrain growth; secondary outcomes were postmortem histopathology, brain and intestinal transcriptomics, and plasma metabolomics. Results: Among 179 infants with term-equivalent MRI (89 NEC, 90 controls; 102 [57.0%] male; mean [SD] gestational age, 26.3 [2.2] weeks), NEC was associated with a higher prevalence of pontine abnormalities (12.4% vs 1.1%; adjusted prevalence ratio, 9.14; 95% CI, 1.16–71.92), alongside white matter, callosal, and cerebellar abnormalities. In postmortem tissue (n = 27), white matter pathology qualitatively resembled that of prematurity, whereas the hindbrain showed distinct regional phenotypes. The cerebellum exhibited external granular layer contraction (42% reduction; P = .04), while the pons showed neuronal injury with increased morphological apoptotic burden (P ≤ .05). Brain and intestinal transcriptomics (n = 14), complemented by plasma metabolomics (n = 29), revealed divergent inflammatory and metabolic signatures. The pons showed increased neuroinflammatory and reduced mitochondrial programs, whereas the cerebellum showed reduced metabolic and cell-cycle programs alongside circulating lysophospholipid depletion. Serial ultrasonography (n = 159), anchored to the cohort mean NEC onset age of 29 weeks’ postmenstrual age, identified earlier cerebellar dimensional deficits (P = .015) and later pontine growth deceleration ( P < .001). Conclusions and Relevance: In this cohort study, NEC-associated hindbrain pathology showed asynchronous growth patterns accompanied by distinct cellular and molecular phenotypes. These findings support investigation of regional vulnerability and stage-specific neuroprotection; individual injury timing and causal mechanisms remain unresolved. Key Points: Question: How does necrotizing enterocolitis (NEC) affect specific brain regions across the clinical course of disease in preterm infants? Findings: In this multimodal cohort study of 253 preterm infants, NEC was associated with asynchronous hindbrain abnormalities. Cerebellar dimensional deficits were detectable before the cohort mean age at NEC onset and persisted without detectable further slowing, alongside progenitor layer contraction and reduced growth-related programs. Pontine growth deceleration emerged after the mean onset age with neuronal injury and inflammatory transcriptomic enrichment. Meaning: Regional differences in growth, cellular pathology, and molecular programs support investigation of stage-specific neuroprotective approaches to NEC-associated brain injury.

Intrinsic programs shape brain maturation; however, which of these programs are perturbed by prematurity as well as the molecular pathways involved remain unknown. The human cerebellum serves as a paradigm of extrauterine development—its accelerated growth and circuit formation align with the third trimester, a period disrupted by preterm delivery. Using multimodal datasets encompassing in vivo neuroimaging and neurodevelopmental outcomes with postmortem spatial-transcriptomic and histopathological profiling, we show that prematurity diverts the cerebellar developmental trajectory. Cerebellar growth and functional outcomes scaled with gestational age, despite modifying perinatal exposures. Spatially resolved developmental programs underlying macroscopic trajectories were marked by incomplete granule cell maturation and impaired Purkinje cell structural refinement, which indicate lineage-specific developmental asynchrony. Thus, prematurity constitutes biologically displaced maturation, in which infants of equivalent postmenstrual age occupy divergent developmental states.

Background: Effective autoresuscitation during hypoxic stress demands the cerebellum integrate autonomic and arousal responses. It remains unknown if the SIDS cerebellum possesses the metabolic stability and developmental maturity to sustain this function. We aimed to define if cerebellar dysfunction constitutes a silent failure point within the central respiratory network. Methods: We performed multi-omic integration of RNA-sequencing, targeted metabolomics, and quantitative histology on postmortem cerebellar tissue from SIDS infants and age-matched controls. Cross-omic concordance analysis mapped the relationship between metabolite abundance and the transcriptional directionality of corresponding metabolic enzymes. Results: The SIDS cerebellum revealed a distinct profile defined by neuroinflammation and altered GABA and glutamate metabolism. Targeted metabolomics confirmed a metabolic imbalance with significantly elevated glutamate and GABA ( P < 0.05). Quantitative histology exposed concurrent developmental arrest; persistent external granule layers highlighted morphological immaturity, explaining the failure to mount effective compensatory responses. Conclusions: Metabolic pathology in the SIDS cerebellum is intrinsically linked to structural immaturity. This developmental arrest creates a compromised environment lacking the metabolic competence to support neural homeostasis, preventing critical arousal responses required for survival. Impact: Multi-omic and neuropathological analysis reveal that structural immaturity in SIDS cerebellum is directly linked to neuroinflammation and metabolic excitotoxicity. Developmental arrest leaves the tissue metabolically incompetent to support the neural homeostasis required for infant survival during hypoxic stress. This study shifts the focus of SIDS pathology beyond traditional brainstem mechanisms by establishing the cerebellum as a critical, point of failure within the central respiratory network, providing a direct link between morphological delay and functional neurochemical disruption These findings offer translatable paradigm for understanding SIDS vulnerability and identify specific metabolic targets for future diagnostic risk screening and therapeutic interventions.

Performing live-cell microscopy and high-dimensional gene expression measurements on the same cells is crucial for unraveling the molecular mechanisms underlying complex temporal phenotypes, yet this remains challenging using traditional approaches. To address these limitations, we developed Video Imaging with Spatial-Temporal Analysis by FISH (VISTA-FISH), a technique that matches live-cell recordings of cultured cells with the expression of thousands of genes in the same cell at the end of the video. Moreover, VISTA-FISH can simultaneously detect CRISPR guide RNAs in pooled screens, enabling investigation into the fundamental underpinnings of cellular activity, organelle transport, and other pivotal cellular functions. Using VISTA-FISH, we measured gene expression and neuron activity in the same differentiating neurons. This combined single-cell transcriptomic and video data allowed us to link differentiation stage, subcellular transcript localization, and cell subtype with neuron activity. Using these measurements, we built a model to predict activity from expression. Finally, we performed a pooled CRISPR interference screen with live-cell lysosome imaging, identifying alterations in lysosome movement and morphology as well as gene expression in perturbed neurons. Collectively, these studies position VISTA-FISH as a powerful new tool for elucidating the molecular mechanisms underlying neuron activity, organelle trafficking, and other complex temporal phenotypes.

Intrinsic programs govern brain maturation, but whether preterm birth induces a persistent shift in these processes remains unknown. The human cerebellum provides a model of extrauterine development; its most rapid growth and circuit maturation occur during the third trimester, a period disrupted by early delivery. By leveraging multimodal in vivo and postmortem tissue analyses, we show that preterm birth constrains cerebellar maturation. Cerebellar growth and functional outcomes scaled with gestational age at birth, paralleled by cellular and spatial molecular programs marking incomplete granule cell maturation and impaired Purkinje cell structural refinement. These lineage-specific alterations indicate developmental asynchrony in prematurity. Thus, in preterm infants, gestational age at birth establishes a cerebellar maturational ceiling, such that postnatal age does not correspond to biological maturity.

Preterm survivors often develop motor and socio-cognitive impairments, yet mechanisms remain limited because prematurity arises from diverse combinations of perinatal insults. Here, we use a mouse model with experiment-controlled timing and sequence of maternal immune activation (MIA) and neonatal hypoxia (Hx), two of the most consequential prematurity-associated insults. We show that these insults remodel the developing cerebellum through distinct processes. Hx arrests granule cell maturation and reduces presynaptic excitatory input to Purkinje cells, whereas MIA perturbs granule cell migration and Purkinje dendritic development. Sequential MIA followed by Hx results in progressive neuronal mitochondrial damage and a reprogrammed circuit state. Behavioral phenotyping links these circuit states to alterations in motor-learning and coordination, translating into divergent sensorimotor organization during social investigation. These results identify developmental sequencing following preterm-birth insults as a determinant of cerebellar resilience, providing a mechanistic framework for heterogeneity in neurodevelopmental outcomes after preterm birth.

Hypothermia (HT) is used clinically for neonatal hypoxic–ischemic encephalopathy (HIE); however, the brain protection is incomplete and selective regional vulnerability and lifelong consequences remain. Refractory damage and impairment with HT cooling/rewarming could result from unchecked or altered persisting cell death and proteinopathy. We tested two hypotheses: (1) HT modifies neurodegeneration type, and (2) intrinsically disordered proteins (IDPs) and encephalopathy cause toxic conformer protein (TCP) proteinopathy neonatally. We studied postmortem human neonatal HIE cases with or without therapeutic HT, neonatal piglets subjected to global hypoxia-ischemia (HI) with and without HT or combinations of HI and quinolinic acid (QA) excitotoxicity surviving for 29–96 h to 14 days, and human oligodendrocytes and neurons exposed to QA for cell models. In human and piglet encephalopathies with normothermia, the neuropathology by hematoxylin and eosin staining was similar; necrotic cell degeneration predominated. With HT, neurodegeneration morphology shifted to apoptosis-necrosis hybrid and apoptotic forms in human HIE, while neurons in HI piglets were unshifting and protected robustly. Oligomers and putative TCPs of α-synuclein (αSyn), nitrated-Syn and aggregated αSyn, misfolded/oxidized superoxide dismutase-1 (SOD1), and prion protein (PrP) were detected with highly specific antibodies by immunohistochemistry, immunofluorescence, and immunoblotting. αSyn and SOD1 TCPs were seen in human HIE brains regardless of HT treatment. αSyn and SOD1 TCPs were detected as early as 29 h after injury in piglets and QA-injured human oligodendrocytes and neurons in culture. Cell immunophenotyping by immunofluorescence showed αSyn detected with antibodies to aggregated/oligomerized protein; nitrated-Syn accumulated in neurons, sometimes appearing as focal dendritic aggregations. Co-localization also showed aberrant αSyn accumulating in presynaptic terminals. Proteinase K-resistant PrP accumulated in ischemic Purkinje cells, and their target regions had PrP-positive neuritic plaque-like pathology. Immunofluorescence revealed misfolded/oxidized SOD1 in neurons, axons, astrocytes, and oligodendrocytes. HT attenuated TCP formation in piglets. We conclude that HT differentially affects brain damage in humans and piglets. HT shifts neuronal cell death to other forms in human while blocking ischemic necrosis in piglet for sustained protection. HI and excitotoxicity also acutely induce formation of TCPs and prion-like proteins from IDPs globally throughout the brain in gray matter and white matter. HT attenuates proteinopathy in piglets but seemingly not in humans. Shifting of cell death type and aberrant toxic protein formation could explain the selective system vulnerability, connectome spreading, and persistent damage seen in neonatal HIE leading to lifelong consequences even after HT treatment.

Neonatal hypoxia (Hx) causes white matter (WM) injury, particularly in the cerebellum. We previously demonstrated that Hx-induced reduction of cerebellar Purkinje cell (PC) activity results in locomotor deficits. Yet, the mechanism of Hx-induced cerebellar WM injury and associated locomotor abnormalities remains undetermined. Here, we show that the cerebellar WM injury and linked locomotor deficits are driven by PC activity and are reversed when PC activity is restored. Using optogenetics and multielectrode array recordings, we manipulated PC activity and captured the resulting cellular responses in WM oligodendrocyte precursor cells and GABAergic interneurons. To emulate the effects of Hx, we used light-activated halorhodopsin targeted specifically to the PC layer of normal mice. Suppression of PC firing activity at P13 and P21 phenocopied the locomotor deficits observed in Hx. Moreover, histopathologic analysis of the developing cerebellar WM following PC inhibition (P21) revealed a corresponding reduction in oligodendrocyte maturation and myelination, akin to our findings in Hx mice. Conversely, PC stimulation restored PC activity, promoted oligodendrocyte maturation, and enhanced myelination, resulting in reversed Hx-induced locomotor deficits. Our findings highlight the crucial role of PC activity in cerebellar WM development and locomotor performance following neonatal injury.

In critically ill newborns, exposure to hypercapnia (HC) is common and often accepted in neonatal intensive care units to prevent severe lung injury. However, as a “safe” range of arterial partial pressure of carbon dioxide levels in neonates has not been established, the potential impact of HC on the neurodevelopmental outcomes in these newborns remains a matter of concern. Here, in a newborn Yorkshire piglet model of either sex, we show that acute exposure to HC induced persistent cortical neuronal injury, associated cognitive and learning deficits, and long-term suppression of cortical electroencephalogram frequencies. HC induced a transient energy failure in cortical neurons, a persistent dysregulation of calcium-dependent proapoptotic signaling in the cerebral cortex, and activation of the apoptotic cascade, leading to nuclear deoxyribonucleic acid fragmentation. While neither 1 h of HC nor the rapid normalization of HC was associated with changes in cortical bioenergetics, rapid resuscitation resulted in a delayed onset of synaptosomal membrane lipid peroxidation, suggesting a dissociation between energy failure and the occurrence of synaptosomal lipid peroxidation. Even short durations of HC triggered biochemical responses at the subcellular level of the cortical neurons resulting in altered cortical activity and impaired neurobehavior. The deleterious effects of HC on the developing brain should be carefully considered as crucial elements of clinical decisions in the neonatal intensive care unit.